‘Programmed cell death’ or apoptosis is a key event in multicellular organisms, defining a genetically encoded cell death program which is morphologically, biochemically and molecularly distinct from necrosis (Vermes et al., J Immunol Meth., (2000) 243, 167-190). The characteristic morphological signs of apoptosis (cellular shrinkage, membrane blebbing, nuclear condensation and fragmentation) are the results of a complex biochemical cascade of events which is an integral part of physiological homeostasis.
Apoptosis ensures an equilibrium between cell proliferation and cell death, thus playing a regulatory role in the control of the size of cell populations and tissues. Aberrations in cell death signalling, in membrane or cytoplasmic receptors, or alterations in genes that govern apoptosis are involved in the pathogenesis of congenital malformations and many acquired diseases (Haanen & Vermes, Eur J. Obstetr. Gynecol., (1996) 64, 129-133). Too little apoptosis may result in malignancies (Tomlinson & Bodmer, Proc. Natl. Acad. Sci. USA, (1995) 92, 11130-11134), Leukemias (Sachs, Proc. Natl. Acad. Sci. USA, (1996) 93, 4742-4749) or the resistance to anticancer therapy (Pahor et al., Lancet, (1996) 348, 493-497). Too much apoptosis can result in immune deficiency (Meyaard et al., Science (1992) 257, 217-219) and degenerative conditions (Griffith et al., Science, (1995) 270, 1189-1192).
There is therefore considerable interest within the medical, pharmaceutical and toxicological sciences in developing a greater understanding of the events which trigger and regulate apoptosis. Furthermore, there is a need to develop new techniques which can be used to identify, quantify and characterise agents which can modulate this phenomenon.
Assays for Detecting Apoptosis
A large number of assays have been developed to detect the onset of programmed cell death (Sgone & Wick, Int Arch Allergy Immunol., (1994) 105, 327-332; Sgone & Gruber, Exp Gerontol., (1998) 33, 525-533). These assays are based upon a wide range of events associated with cell death and have traditionally included light and electron microscopy with vital staining and nuclear dyes. Biochemical methods are often employed, for example based upon DNA laddering or degradation, DNA end labelling techniques (e.g. TUNEL—terminal deoxynucleotide transferase dUTP Nick End labelling), nuclease activity and lactate dehydrogenase enzyme release.
Flow cytometry tends to be the most widely used method (Vermes et al., J Immunol Methods, (2000) 243, 167-190) for detecting and quantifying apoptosis because it is amenable to screening large numbers of cells. This fluorescence—based technique employs vital dyes, antibodies to apoptotic enzymes (e.g. caspases) and single stranded DNA breaks, together with probes for measuring calcium flux and phospholipid redistribution.
Flow cytometry allows in vivo analysis of cells in suspension, one at a time, at rates of 1000 to 10,000 cells/s. However, one problem with flow cytometry is that it can only be used in cells in suspension, such as liquid cell cultures and cells derived from the hemopoietic system. Furthermore flow cytometry of tissue cells requires physical and enzymatic manipulation to get the cells in suspension, which by itself may trigger apoptosis, necessitating checks by conventional light or fluorescence microscopy.
Existing methods are thus based upon late events in apoptosis (e.g. DNA degradation, caspase assays) and many require cellular fixation and staining with specific antibodies/dyes. None of the techniques described above provide a homogeneous living cell assay, based upon the early events in apoptosis, in real time nor discriminate from cellular necrosis. Furthermore, none of these assays are amenable to high throughput live cell screening which is required to test large numbers of compounds for their ability to modulate apoptosis.
Cytochrome c Translocation as a Marker for Apoptosis
Cytochrome c is a nuclear encoded protein which is targeted to the mitochondria where it performs its biological function as an electron carrier. The translocation of cytochrome c from the mitochondria to the cytoplasm in response to apoptotic stimuli is an early and critical step in the commitment of the cell to undergo apoptosis (Li et al., Cell (1997) 91, 479-489). Cytochrome c binds strongly to apoptosis protease activation factor-1 (Apaf-1) in the cytosol (Zou et al., Cell (1997) 90, 405-413). In the presence of cofactors the resulting cytochrome c: Apaf-1 assembles into a multimeric ‘apoptosome’ that binds and activates a protease zymogen, procaspase-9 (Srinivasula et al., Mol. Cell (1998) 1, 949-957). This results in the activation of the ‘caspase cascade’ whereby many intracellular substrates are cleaved disabling important cellular processes and breaking down structural components of the cell (Slee et al., J Cell. Biol. (1999) 144, 281-292; Skulachev, FEBS Lett., (1998) 423, 275-280). A schematic diagram illustrating the mitochondrial role in apoptosis is shown in FIG. 1.
Recent in vitro studies (Kluck et al., J. Biol. Chem., (2000), 275, 16127-16133; Yu et al., J. Biol. Chem., (2001), 276, 1304-13038) have identified the molecular determinants involved in the cytochrome c: Apaf-1 interaction. Horse cytochrome c has been shown to be highly homologous to human cytochrome c and can initiate caspase activation, whereas yeast cytochrome c did not measurably bind to Apaf-1 nor activate caspase (Yu et al., J Biol Chem., (2001) 276, 13034-13038). These studies focussed on the key differences between horse and yeast cytochrome c and used site directed mutagenesis to generate mutant variants which were subsequently analysed with respect to their ability to activate caspase. The results from this in vitro work indicated that residue 7, 25, 39, 62-65 and 72 were critical amino acids for cytochrome c:Apaf-1 interaction. Notably the mutation K72A showed no detectable binding or caspase-9 activation. While the mutation of lysine 72 to alanine abolished the interaction between cytochrome c and Apaf1, respiratory function of cytochrome c was unaffected.
Although the authors demonstrated reduced binding to Apaf-1 and caspase-9 activation in vitro it is not known whether the cytochrome c mutant proteins would behave in a similar manner in living cells, targeting the mitochondria and not inducing apoptosis.
Recent reports by Abdullaev et al. (Biochem J. (2002) 362, 749-754), again based on in vitro experiments, indicate that the horse K72 mutants described by Yu et al. (J Biol Chem., (2001) 276, 13034-13038) show the same level of caspase activation as the wild-type protein if present at 2-12 fold higher concentrations than the wild type protein. In contrast, the authors reported that a horse K4E cytochrome c mutant was inactive in activating caspase and conclude that this mutant, rather than K72 mutants, would be a good candidate for in vivo knock-in studies on the role of cytochrome c in apoptosis.
Fluorescent Proteins
The use of Green Fluorescent Protein (GFP) derived from Aequorea victoria is now well known for research into many cellular and molecular-biological processes. Cytochrome c-GFP fusions have been used in studies on apoptosis. Thus Heiskanen et al. (J Biol Chem., (1999) 274, 5654-5658) expressed a cytochrome c-GFP fusion, based upon rat cytochrome c, in rat pheochromocytoma-6 (PC6) cells. Induction of apoptosis by staurosporine led to release of the fusion from the mitochondria which was accompanied by mitochondrial depolarisation.
Goldstein et al., (Nat Cell Bio., (2000) 2, 156-160) demonstrated mitochondrial localisation of a cytochrome c-GFP fusion, based upon human cytochrome c, over-expressed in HeLa cells. A range of apoptotic inducers were shown to cause rapid release of cytochrome c-GFP.
Other researchers have also used cells which have been transiently transfected to over-produce cytochrome c-GFP fusion proteins to investigate apoptosis. Thus Goa et al., (J. Cell Sci., (2001) 114, 2855-2862) monitored the dynamic redistribution of GFP-tagged cytochrome c and the morphological changes of mitochondria within living HeLa cells during UV-induced apoptosis. Similarly, Lim et al., (J Biomed Sci (2002); 9, 488-506) explored the biochemical basis of cytochrome c-GFP localisation by transiently transfecting COS-7 cells with various GFP constructs and determining sub-cellular distribution using fluorescence and immunochemical techniques.
Problem to be Addressed
There is a need to develop sensitive assays which are amenable to high throughput screening in living cells and which allow detection and analysis in real time of the early events in apoptosis.
While over-expression of cytochrome c in cells has been demonstrated, the resulting high levels of protein lead to apoptosis and cell death, necessitating the use of complex inducible/transient systems for studies involving up-regulation of cytochrome c levels (Chandra et al., J Biol Chem. (2002) 277, 50842-50854). To date, it has not been possible to generate stable cell lines over-expressing this protein which could be reliably used for screening purposes.
There is thus a need for a cytochrome c protein which, when coupled to a reporter group to form a fusion construct, can be expressed in cells to provide stable cell lines. The resulting construct can act as a biosensor within the cells for early events in the induction or repression of apoptosis. Stable cell lines overexpressing such constructs are suitable for high throughput screening purposes to identify agents which modulate apoptosis.
The present invention addresses this problem and provides a fusion construct which has no observable toxicity to cells and acts as a biosensor to permit the detection of early events in apoptosis in living cells. The stably transformed cells of the invention can still undergo apoptosis due to the presence of endogenous cytochrome c, thus allowing detection and determination of a second apoptotic signaling event whether up or downstream of the cytochrome c translocation.